Use of Chemical Chelators as Reversal Agents for Drug-Induced Neuromuscular Block

ABSTRACT

The invention relates to the use of chemical chelators for the preparation of a medicament for the reversal of drug-induced neuromuscular block, to a kit for providing neuromuscular block and its reversal, and to cyclophane derivatives having the general formula A 
     
       
         
         
             
             
         
       
     
     wherein R is
 
(CH 2 ) 5 ,
 
     
       
         
         
             
             
         
       
     
     or 
     
       
         
         
             
             
         
       
     
     or
 
the general formula B
 
     
       
         
         
             
             
         
       
     
     wherein X is
         (CH 2 ) 5 ,       

     
       
         
         
             
             
         
       
     
     or 
     
       
         
         
             
             
         
       
     
     or a pharmaceutically acceptable salt thereof.

The invention relates to the use of chemical chelators for thepreparation of a medicament for the reversal of drug-inducedneuromuscular block, and to a kit for providing neuromuscular block andits reversal.

A neuromuscular blocking agent (NMBA, also called a muscle relaxant) isroutinely used during the administration of anaesthesia to facilitateendotracheal intubation and to allow surgical access to body cavities,in particular the abdomen and thorax, without hindrance from voluntaryor reflex muscle movement. NMBAs are also used in the care ofcritically-ill patients undergoing intensive therapy, to facilitatecompliance with mechanical ventilation when sedation and analgesia alonehave proved inadequate.

Based on their mechanisms of action, NMBAs are divided into twocategories: depolarizing and non-depolarizing. Depolarizingneuromuscular blocking agents bind to nicotinic acetylcholine receptors(nAChRs) at the neuromuscular junction in a way similar to that of theendogenous neurotransmitter acetylcholine. They stimulate an initialopening of the ion channel, producing contractions known asfasciculations. However, since these drugs are broken down onlyrelatively slowly by cholinesterase enzymes, compared to the very rapidhydrolysis of acetylcholine by acetylcholinesterases, they bind for amuch longer period than acetylcholine, causing persistent depolarizationof the end-plate and hence a neuromuscular block. Succinylcholine(suxamethonium) is the best known example of a depolarizing NMBA.

Non-depolarizing neuromuscular blocking agents compete withacetylcholine for binding to muscle nAChRs, but unlike depolarizingNMBAs, they do not activate the channel. They block the activation ofthe channel by acetylcholine and hence prevent cell membranedepolarization, and as a result, the muscle will become flaccid. Mostclinically-used NMBAs belong to the non-depolarizing category. Theseinclude tubocurarine, atracurium, (cis)atracurium, mivacurium,pancuronium, vecuronium, rocuronium and rapacuronium (Org 9487).

At the end of surgery or a period of intensive care, a reversal agent ofNMBAs is often given to the patient to assist the recovery of musclefunction. Most commonly used reversal agents are inhibitors ofacetylcholinesterase (AChE), such as neostigmine, edrophonium andpyridostigmine. Because the mechanism of action of these drugs is toincrease the level of acetylcholine at the neuromuscular junction byinhibiting the breakdown of acetylcholine, they are not suitable forreversal of depolarizing NMBAs such as succinylcholine. The use of AChEinhibitors as reversal agents leads to problems with selectivity, sinceneurotransmission to all synapses (both somatic and autonomic) involvingthe neurotransmitter acetylcholine is potentiated by these agents. Thisnon-selectivity may lead to many side-effects due to the isnon-selective activation of muscarinic and nicotinic acetylcholinereceptors, including bradycardia, hypotension, increased salivation,nausea, vomiting, abdominal cramps, diarrhoea and bronchoconstriction.Therefore in practice, these agents can be used only after or togetherwith the administration of atropine (or glycopyrrolate) to antagonizethe muscarinic effects of acetylcholine at the muscarinic receptors inthe autonomic parasympathetic neuro-effector junctions (e.g. the heart).The use of a muscarinic acetylcholine receptor (mAChR) antagonist suchas atropine causes a number of side-effects, e.g., tachycardia, drymouth, blurred vision, and furthermore may affect cardiac conduction.

A further problem with anticholinesterase agents is that residualneuromuscular activity must be present (>10% twitch activity) to allowthe rapid recovery of neuromuscular function. Occasionally, either dueto hyper-sensitivity of the patient or accidental overdose,administration of NMBAs can cause complete blockade of neuromuscularfunction (“deep block”). At present, there is no reliable treatment toreverse such a ‘deep block’. Attempts to overcome a ‘deep block’ withhigh doses of AChE inhibitors has the risk of inducing a “cholinergiccrisis”, resulting in a broad range of symptoms related to enhancedstimulation of nicotinic and muscarinic receptors.

There is thus a need for an alternative method for reversing the actionof NMBAs, i.e. to restore the muscular contractions.

The present invention provides for the use of chemical chelators (orsequestrants) as reversal agents. In one aspect the invention pertainsto the use of a chemical chelator capable of forming a guest-hostcomplex for the manufacture of a medicament for the reversal ofdrug-induced neuromuscular block.

The use of chemical chelators as reversal agents for NMBAs has theadvantage that they are effective in reversing the action of bothdepolarizing and non-depolarizing NMBAs, since chemical chelators do notcompete with the NMBA for binding to nAChRs. Their use does not increasethe level of acetylcholine and therefore they produce fewer side effectsthan AChE-based reversal agents. In addition, there is no need for thecombined use of a AChE inhibitor and a mAChR antagonist (e.g.,atropine). The chemical chelators of the invention may further be safelyemployed for the reversal of ‘deep block’.

The term chemical chelator (or sequestrant), as used in the presentinvention, means any organic compound which can engage in host-guestcomplex formation with a neuromuscular blocking agent. The chemicalchelator acts as the host molecule, the neuromuscular blocking agentbeing the guest molecule. The specific molecular complex, the guest-hostcomplex, is defined as an organized chemical entity resulting from theassociation of two or more components held together by noncovalentintermolecular forces.

The chemical chelators (or sequestrants), according to the invention,are host molecules selected from various classes of, mostly cyclic,organic compounds which are known for their ability to form inclusioncomplexes with various organic compounds in aqueous solution, e.g.cyclic oligosaccharides, cyclophanes, cyclic peptides, calixarenes,crown ethers and aza crown ethers. Formation of inclusion complexes(also called encapsulation, or chemical chelation) is part of thewell-known area of ‘supramolecular chemistry’ or ‘host-guest chemistry’.Many cyclic organic compounds are known to be capable of forming aninclusion complex with another molecule, organic or inorganic. Thestructures and chemistry of these compounds are well documented(Comprehensive Supramolecular Chemistry, Volumes 1-11, Atwood J. L.,Davies J. E. D., MacNicol D. D., Vogtle F., eds; Elsevier Science Ltd.,Oxford, UK, 1996).

Preferred chemical chelators for use with the present invention arecyclic oligosaccharides, cyclophanes and calixarenes.

Examples of cyclic oligosaccharides suitable for use with the inventionare the cyclodextrins, a category of naturally occurringcyclomaltooligosaccharides, the cyclomannins (5 or moreα-D-mannopyranose units linked at the 1,4 positions by α linkages), thecyclogalactins (5 or more β-D-galactopyranose units linked at the 1,4positions by β linkages), the cycloaltrins (5 or more α-D-altropyranoseunits linked at the 1,4 positions by α linkages), each of which arecapable of forming guest-host complexes. Cyclic oligosaccharides ofdifferent monosaccharide compositions, accessible through total chemicalsynthesis, represent further chemical chelators capable of interactionwith a neuromuscular blocking agent. For example,cyclo-[(1-4)-α-L-rhamno-pyranosyl-(1-4)-α-D-mannopyranosyl]tetraoside,was found to be effective in reversal of the action of the neuromuscularblocking agent rocuronium bromide.

A particularly preferred class of cyclic oligosaccharide chelatorsaccording to the invention is formed by the cyclodextrins:

Cyclodextrins are cyclic molecules containing six or moreα-D-glucopyranose units linked at the 1,4 positions by a linkages as inamylose. As a consequence of this cyclic arrangement, the cyclodextrinsexist as conical shaped molecules with a lipophilic cavity which canattract guest molecules whilst the outside is more hydrophilic andwater-soluble. Cyclodextrins composed of six, seven, eight and nineglucopyranose units are commonly known as α-, β-, γ- andδ-cyclodextrins, respectively.

Both the native cyclodextrins (α, β, γ) which are prepared by enzymaticdegradation of starch, and especially a number of chemically modifiedforms thereof, have already found, by virtue of their ability to formguest-host complexes, numerous applications, especially in thepharmaceutical field. Stella and Rajewski (Pharmaceutical Research, 14,556-567, 1997) have recently reviewed pharmaceutical applications of thecyclodextrins. The major is applications are in the pharmaceuticalformulations of drugs in order to solubilize and/or to stabilize a drugfor oral, nasal, ophthalmic, dermal, rectal and parenteraladministration.

The term cyclodextrin as used in relation to the present inventionincludes both the native cyclodextrins and chemically modified formsthereof.

An overview on such chemically modified cyclodextrins as drug carriersin drug delivery systems is described by Uekama et al. (Chemical Reviews1998, 98, 2045-2076). Chemical modification of cyclodextrins can be madedirectly on the native α-, β- or γ-cyclodextrin rings by reacting achemical reagent (nucleophiles or electrophiles) with a properlyfunctionalised cyclodextrin (for an recent overview of methods for theselective modification of cyclodextrins see Khan A. R. et al. Chem. Rev.1998, 98, 1977-1996). To date, more than 1,500 cyclodextrin derivativeshave been made by chemical modification of native cyclodextrins(Jicsinszky L. et al Comprehensive Supramolecular Chemistry, Volume 3.Cyclodextrins, Atwood J. L., Davies J. E. D, MacNicol D. D., Vogtle F.,eds; Elsevier Science Ltd., Oxford, UK, 1996, pp 57-188).

Many direct modifications of a native cyclodextrin result in a mixtureof isomers without precisely defined positions of substitution. Such amixture of positional isomers is often referred to as a statisticmixture, the number of substituents attached at each cyclodextrinmolecule in such a statistic mixture being expressed as the averagedegree of substitution (DS). Most cyclodextrin derivatives studied forpharmaceutical applications are statistic mixtures (Szente L. andSzejtli J., Adv. Drug Delivery Rev. 1999, 36, 17-28). Directmodification of a cyclodextrin does not alter the constitution or theconfiguration of the repeating α-D-glucopyranosyl units.

Cyclodextrins can also be prepared by de novo synthesis, starting withglucopyranose (Gattuso G. et al Chem. Rev. 1998, 98, 1919-1958). In thisway, one can prepare not only the naturally occurring cyclic(1→4)-linked cyclodextrins but also the cyclic (1→3)-, (1→2)-, and(1→6)-linked oligopyranosides. Such a synthesis can be accomplished byusing various chemical reagents or biological enzymes such ascyclodextrin transglycosylase. By using different sugar units as thestarting materials, one can thereby prepare various homogeneous orheterogeneous cyclic oligosaccharides. Chemical modification ofcyclodextrins is thus known to modulate their properties and can be usedfor the design of reversal agents selective for a specific neuromuscularblocking agent.

It will be clear to the skilled person that for a particularneuromuscular blocking agent a chemical chelator host can be developedhaving a hydrophobic cavity of a shape and size adapted to the guestmolecule, while in addition to the hydrophobic interactions between thehost and the guest charge interactions can be of importance for complexformation. Since the chemical chelators of the invention are forparenteral application they will have to be water-soluble. A specifichost molecule can be designed and prepared to contain functionalitiescomplementary to those of the guest molecule in such a manner that itresults in maximum intermolecular interaction via for examplehydrogen-bond, hydrophobic, electrostatic, van der Waals, and π-πinteractions. Thus, for example, for a guest molecule containing basicfunctional groups or positive charge, a host molecule containing acidicfunctional groups or negative charge could be made to increase ionicinteraction between the guest and the host. When such a host-guestcomplex is formed via inclusion or partial inclusion, the cavity size ofthe host molecule is also very important

The interaction between a chemical chelator and a neuromuscular blockingagent can be analyzed by physical methods such as nuclear magneticresonance spectroscopy (nmr) and microcalorimetry.

The most preferred cyclodextrins for use in the invention areγ-cyclodextrin and derivatives thereof.

Many of the commonly used neuromuscular blocking agents, or musclerelaxants, such as rocuronium, pancuronium, vecuronium, mivacurium,atracurium, (cis)atracurium, succinylcholine and tubocurarine, arecompounds having 1 or 2 cationic sites when in neutral aqueous medium.Cyclodextrins having anionic sites in their structure are among thepreferred chemical chelators according to the invention.

The preference for anionic chemical chelators for the reversal of theabove mentioned neuromuscular blocking agents also applies for chemicalchelators of the invention which belong to the cyclophanes.

Cyclophanes are a class of bridged aromatic compounds which havewell-defined hydrophobic inclusion cavities constructed by aromaticrings incorporated in their macrocyclic structures. By introducing polarand hydrophilic functional groups such as hydroxyls and carboxyls intothe artificial host compounds, cyclophanes can be made water-soluble andsuitable for forming inclusion complex in aqueous media (Vogtle F. etal. Comprehensive Supramolecular Chemistry, Volume 2. Molecularrecognition: Receptors for molecular guests, Atwood, J. L., Davies, J.E. D., MacNicol, D. D., Vogtle, F., eds; Elsevier Science Ltd., Oxford,UK, 1996, pp 211-266). Water soluble anionic cyclophanes are describedby Miyake et al. (Tetr. Letters 32, 7295-7298, 1991; Chem. Pharm. Bull.41, 1211-1213, 1993) as hosts for cationic aromatic guests. Analogously,cationic cyclophanes were shown to form inclusion complexes in aqueoussolution with anionic and neutral aromatic compounds.

In a preferred embodiment of the invention the chemical chelator ischosen from cyclophanes having the general formula A

is wherein R is

-   -   (CH₂)₅,

or

and n is 1-5; or the general formula B

wherein X is

-   -   (CH₂)₅,

or

The compound according to formula A, wherein R is CH₂-phenyl-CH₂ and nis 2, i.e.N,N′,N″,N″′-tetrakis(3-carboxypropionyl)-3,4,5,6,7,8,26,27,28,29,30,31-dodecahydro-1,10,24,33-tetraaza[2.2.1.2.2.1]paracyclophane(compound 23), and the compound according to formula B wherein X isCH₂-phenyl-CH₂, i.e.1,10,24,33-tetraoxa-12,20,35,43-tetracarboxy-[2.2.1.2.2.1]-paracyclophane(compound 18 is a preferred cyclophane derivative to reverse the actionof rocuronium bromide.

In a further aspect the invention provides for novel paracyclophanederivatives having formula A or Formula B, as defined above, orpharmaceutically acceptable salts thereof. Examples of such salts arethe potassium, sodium and ammonium salts and the like.

The chemical chelators for use in the invention are administeredparenterally. The injection route can be intravenous, subcutaneous,intradermal, intramuscular, or intra-arterial. The intravenous route isthe preferred one. The exact dose to be used will necessarily bedependent upon the needs of the individual subject to whom themedicament is being administered, the degree of muscular activity to berestored and the judgement of the anaesthetist/critical-care specialist.Extracorporal application of the chemical chelators of the invention,for instance by mixing of the chemical chelator with the blood duringdialysis or during plasmapheresis, is also contemplated.

In a further aspect the invention relates to a kit for providingneuromuscular block and its reversal comprising (a) a neuromuscularblocking agent, and (b) a chemical chelator capable of forming aguest-host complex with the neuromuscular blocking agent. With a kitaccording to the invention is meant a formulation, which containsseparate pharmaceutical preparations, i.e. the neuromuscular blockingagent and a chemical chelator, i.e. the reversal agent. The componentsof such a kit of parts are to be used sequentially, i.e. theneuromuscular blocking agent is administered to a subject in needthereof, which is followed, at a point in time when restoration ofmuscle function is required, by the administration of the reversalagent, i.e. a chemical chelator capable of forming a guest-host complexwith the neuro-muscular blocking agent.

A preferred kit, according to the invention, contains a chemicalchelator selected from the group consisting of a cyclic oligosaccharideand a cyclophane, and a neuromuscular blocking agent which is selectedfrom the group consisting of rocuronium, vecuronium, pancuronium,rapacuronium, mivacurium, atracurium, (cis)atracurium, tubocurarine andsuxamethonium. A particularly preferred kit of the invention comprisesrocuronium, as the neuromuscular blocking agent, and γ-cyclodextrin, ora derivative thereof, as the chemical chelator.

Mixed with pharmaceutically suitable auxiliaries and pharmaceuticallysuitable liquids, e.g. as described in the standard reference, Gennaroet al., Remington's Pharmaceutical Sciences, (18th ed., Mack PublishingCompany, 1990, Part 8: Pharmaceutical Preparations and TheirManufacture; see especially Chapter 84 on “Parenteral preparations, pp.1545-1569; and Chapter 85 on “Intravenous admixtures”, pp. 1570-1580)the chemical chelators can be applied in the form of a solution, e.g.for use as an injection preparation.

Alternatively, the pharmaceutical composition may be presented inunit-dose or multi-dose containers, for example sealed vials andampoules, and may be stored in a freeze dried (lyophilised) conditionrequiring only the addition of the sterile liquid carrier, for example,water prior to use.

The invention further includes a pharmaceutical formulation, ashereinbefore described, in combination with packaging material suitablefor said composition, said packaging material including instructions forthe use of the composition for the use as hereinbefore described.

The invention is illustrated in the following examples.

EXAMPLE 1 Cyclodextrin Derivatives

Some of the cyclodextrin derivatives that were employed to demonstratetheir activity as reversal agents according to the invention werecommercially available:

Commercial Source Compound 1: α-cyclodextrin (α-CD) Wacker-Chemie GmbH,Munich, Germany; or ALDRICH Compound 2: carboxymethyl-β-CD (DS = 3.5)Wacker-Chemie GmbH, Munich, Germany sodium salt Compound 3:2-hydroxy-3-trimethylammonio Wacker-Chemie GmbH, Munich, Germanypropyl--β-CD (DS = 3.5) Compound 4: per 2,6-dimethyl--β-CD (DS = 12.6)Wacker-Chemie GmbH, Munich, Germany Compound 5: β-cyclodextrin-phosphatesodium salt (DS = 3) CycloLab, Ltd. Budapest, Hungary Compound 6:β-cyclodextrin-phosphate sodium salt (DS = 8) CycloLab, Ltd. Budapest,Hungary Compound 7: carboxymethyl-β-CD (DS = 3-3.5) CycloLab, Ltd.Budapest, Hungary Compound 8: carboxyethyl-β-CD (DS = 3) CycloLab, Ltd.Budapest, Hungary Compound 9: β-cyclodextrin (β-CD) Wacker-Chemie GmbH,Munich, Germany; or ALDRICH Compound 10: 2-hydroxypropyl-β-CD RBI,Natick, MA 01760, USA Compound 11: γ-cyclodextrin-phospate CycloLab,Ltd. Budapest, Hungary sodium salt (DS = 3) Compound 12:γ-cyclodextrin-phospate CycloLab, Ltd. Budapest, Hungary sodium salt (DS= 7) Compound 13: carboxymethyl-γ-CD (DS = 3.2) CycloLab, Ltd. Budapest,Hungary Compound 14: carboxyethyl-γ-CD (DS = 3.8) CycloLab, Ltd.Budapest, Hungary Compound 15: γ-cyclodextrin (γ-CD) Wacker-Chemie GmbH,Munich, Germany; or FLUKA Compound 16: 2-hydroxypropyl-γ-CD (DS = 4)RBI, Natick, MA 01760, USA

-   -   DS means the degree of substitution, which is the mean number of        hydroxy functions which carry the pertinent substituent.

compounds 2 and 7 are the same, be it from different suppliers.

EXAMPLE 2 Cyclophane Derivatives

Nomenclature: the term paracyclophane refers to a family of compounds inwhich one or more benzene rings are built into a carbocyclic ring systemand in which the p-positions of the benzene rings are part of the ringsystem. The conventional numbering used below for paracyclophane ringsystems is that described by Cram and Abell (J. Am. Chem. Soc. 1955, 77,1179-1186)

A: Tetraaza-paracyclophanes derivatives:

Scheme A depicts the structures of the N-(carboxy)acylated cyclophanederivatives 19-23, which were prepared by acylation of the parentcyclophanes (see Soga T. et al Tetrahedron Lett. 1980, 4351-4, forsynthesis thereof) (1,7,21,27-tetraaza[7.1.7.1]paracyclophane (I),1,10,24,33-tetraaza-[2.2.1.2.2.1]paracyclophane (II) and3,4,5,6,7,8,26,27,28,29,30,31-dodecahydro-1,10,24,33-tetraaza[2.2.1.2.2.1]paracyclophane(III) with the appropriate activated acid derivative.

A1: Compound 21:N,N′,N″,N″′-Tetrakis(3-carboxypropionyl)-1,7,21,27-tetraaza[7.1.7.1]paracyclophane.

To a suspension of 1,7,21,27-tetraaza[7.1.7.1]paracyclophane (400 mg,0.75 mmol) in dichloromethane (5 ml) was added triethylamine (1.05 ml,7.52 mmol) followed by 3-(methoxycarbonyl)propionyl chloride (0.93 ml,7.52 mmol) dissolved in dichloromethane (3 ml). The reaction was stirredunder an atmosphere of nitrogen for 12 h. The reaction was diluted withdichloromethane (20 ml) and washed with water (2×20 ml), dried (MgSO₄)and the solvent removed in vacuo to give a yellow oil, which waspurified by chromatography on silica gel eluting with 5% methanol indichloromethane. The resultant product crystallised on standing. Theproduct was recystallised from chloroform/ether to giveN,N′,N″,N″′-tetrakis[3-(methoxycarbonyl)-propionyl]-1,7,21,27-tetraaza[7.1.7.1]paracyclophane(480 mg, 0.48 mmol, 65%). MS (EI) m/z 989 (M+H)⁺, ¹H NMR (CDCl₃) δ 1.27(m, 4H), 1.45 (m, 5H), 1.64 (m, 3H), 2.25 (t, J 6.8, 8H), 2.56 (t, J6.8, 8H), 3.61 (t, J 7.6, 8H), 3.65 (s, 12H), 4.02 (s, 4H), 7.09 (d, J8.1, 8H), 7.20 (d, J 8.1, 8H); ¹³C NMR (CDCl₃) δ 24.45, 27.85, 29.17,29.37, 40.73, 49.47, 51.68, 128.47, 130.27, 140.15, 140.60, 171.35,173.84.

A mixture of the above tetramethylester (440 mg, 0.45 mmol), potassiumhydroxide pellets (2.51 g, 45 mmol), methanol (9 ml) and water (25 ml)was heated to reflux for 4 h. The reaction was cooled to roomtemperature, most of the solvent removed in vacuo and the residueacidified with 2N HCl. The resultant precipitate was filtered and driedthen recystallised from MeOH/H₂O to give the title compound 21 (142 mg,0.15 mmol, 34%).

MS (EI) m/z 931 (M−H)⁻, ¹H NMR (CD₃OD) δ 1.28 (m, 4H), 1.44 (m, 8H),2.25 (m, 8H), 2.49 (m, 8H), 3.60 (m, 8H), 4.05 (s, 4H), 7.16 (d, J 7.72,8H), 7.30 (d, J 7.72, 8H), ¹³C NMR (CDCl₃) δ 23.95, 27.44, 28.94, 29.11,39.86, 48.62, 128.21, 129.79, 140.01, 140.35, 170.01, 173.07, I.R. (KBr)1736, 1656 cm⁻¹.

In a Similar Manner were Prepared:A2:N,N′,N″,N″′-Tetrakis(carboxyacetyl)-1,7,21,27-tetraaza[7.1.7.1]paracyclophane(Compound 19) starting from 1,7,21,27-tetraaza[7.1.7.1]-paracyclophaneand methyl malonyl chloride.

MS (EI) m/z 877 (M+H)⁺, ¹H NMR (DMSO) δ 1.21 (m, 4H), 1.36 (m, 8H), 2.97(m, 8H), 3.53 (m, 8H), 3.99 (s, 4H), 7.15 (d, J 7.95, 8H), 7.27 (d, J7.95, 8H), 12.40 (s, 4H), ¹³C NMR (DMSO) δ 23.79, 27.19, 39.39, 41.44,48.59, 128.04, 129.83, 145.05, 141.02, 165.69, 169.23, I.R. (KBr) 1736,1625 cm⁻¹.

A3:N,N′,N″,N″′-Tetrakis(4-carboxybutyryl)-1,7,21,27-tetraaza[7.1.7.1]paracyclophane(Compound 20) starting from 1,7,21,27-tetraaza[7.1.7.1]-paracyclophaneand methyl 4-(chloroformyl)butyrate.

MS (EI) m/z 989 (M+H)⁺, ¹H NMR (CD₃OD) δ 1.28 (m, 4H), 1.44 (m, 8H),1.76 (m, 8H), 2.07 (t, J 7.5, 8H), 2.19 (t, J 7.5, 8H), 3.62 (m, 8H),4.05 (s, 4H), 7.11 (d, J 8.17, 8H), 7.27 (d, J 8.17, 8H), ¹³C NMR(CDCl₃) δ 21.86, 25.54, 28.72, 34.02, 34.41, 41.70, 50.60, 129.50,131.47, 141.82, 142.55, 174.69, 177.69, I.R. (KBr) 1736, 1656 cm⁻¹.

A4: N,N′,N″,N″′-Tetrakis(3-carboxypropionyl)1,10,24,33-tetraaza-[2.2.1.2.2.1]paracyclophane (Compound 22) startingfrom cyclophane II and 3-(methoxycarbonyl)propionyl chloride.

¹H NMR (DMSO) δ 2.22 (m, 8H), 2.42 (m, 8H), 3.93 (s, 4H), 4.75 (s, 8H),6.96 (d, J 8.27, 8H), 7.00 (s, 8H), 7.16 (d, J 8.27, 8H), 11.90 (bs,1H), ¹³C NMR (DMSO) δ 28.83, 29.10, 39.93, 51.51, 125.32, 127.83,128.22, 129.43, 136.36, 139.88, 14.00, 170.69, 173.52, I.R. (KBr) 1727,1650 cm⁻¹.

A5:N,N′,N″,N″′-Tetra(3-carboxypropionyl)-3,4,5,6,7,8,26,27,28,29,30,31-dodecahydro-1,10,24,33-tetraaza[2.2.1.2.2.1]paracyclophane(Compound 23) starting from cyclophane III and3-(methoxycarbonyl)propionyl chloride.

MS (EI) m/z 1012 (M−H)⁻, ¹H NMR (DMSO) δ 0.76 (m, 8H), 1.51 (m, 4H),1.67 (m, 8H), 2.15 (m, 8H), 2.36 (t, J 6.60, 8H), 3.37 (s, 8H), 3.97 (s,4H), 7.19 (d, J 8.08, 8H), 7.30 (d, J 8.08, 8H), 11.86 (bs, 4H), ¹³C NMR(DMSO) δ 28.97, 29.09, 36.13, 52.33, 55.59, 128.09, 129.72, 141.55,170.65, 173.52, I.R. (KBr) 1727, 1652 cm⁻¹.

B. Tetraoxa-paracyclophane derivatives.

The ether-linked cyclophanes (compounds 17, 18 and 25) can besynthesised by a ring construction as shown in Scheme B.

The commercially available 5,5′-methylenedisalicylic acid IV wasprotected as methyl ester V which was then alkylated with an appropriatedihalide to yield VI. Reaction of dihalide VI with an equivalentdiphenol IV gave the cyclophanes VII-IX which gave the desiredcarboxylic acid derivatives upon saponification.B1: Compound 17: 1,7,21,27-tetraoxa-9,17,29,37-tetracarboxy[7.1.7.1]paracyclophane.

3,3′Dimethoxycarbonyl-4,4′-dihydroxydiphenylmethane (V)

To methanol (100 ml) saturated with hydrogen chloride gas was added3,3′-dicarboxy-4,4′-dihydroxydiphenylmethane (10 g, 34.69 mmol)portion-wise over 30 min. The mixture was then heated to reflux for 3 h,cooled to room temperature and re-saturated with hydrogen chloride gas.After a further 8 h heating at reflux the solvent was removed in vacuoand the product purified by chromatography on silica gel eluting with25% ethyl acetate/petroleum ether to give the title compound (2.40 g,7.59 mmol, 22%).

¹H NMR (CDCl₃) δ 3.84 (s, 2H), 3.92 (s, 6H), 6.90 (d, J 8.0, 2H), 7.25(dd, J 8.0, 1.0, 2H), 7.63 (d, J 1.0, 2H), 10.65 (s, 2H).

4,4′-Bis(5-bromopentoxy)-3,3′-dicarboxymethyl-4,4′-dihydroxydiphenyl-methane(VIa)

To a stirred suspension of 1,4-dibromopentane (21.8 g, 94.9 mmol) andK₂CO₃ (13.1 g, 94.9 mmol) in dry dimethylformamide (380 ml) at 60° C.,under an atmosphere of nitrogen, was added dropwise a solution of3,3′-dicarboxymethyl-4,4′-dihydroxydiphenylmethane (3.0 g, 9.49 mmol) indry dimethylformamide (190 ml). The resultant mixture was heated afurther 1 h, cooled and filtered. The dimethylformamide was removed invacuo and the product purified by chromatography eluting with 1%methanol in dichloromethane followed by a second purification elutingwith 10% ethyl acetate/heptane to give the title compound (3.51 g, 5.73mmol, 60%).

¹H NMR (CDCl₃) δ 1.60-2.02 (m, 12H), 3.44 (t, J 7.0, 4H), 3.87 (s, 8H),4.01 (t, J 6.5, 4H), 6.87 (d, J 8.0, 2H), 7.22 (dd, J 8.0, 1.0, 2H),7.60 (d, J 1.0, 2H).

1,7,21,27-tetraoxa-9,17,29,37-tetra(methoxycarbonyl)[7.1.7.1]paracyclophane(VII)

A solution of4,4′-bis(5-bromopentoxy)-3,3′-dicarboxymethyl-4,4′-di-hydroxydiphenylmethane(3.51 g, 5.74 mmol) and3,3′-dicarboxymethyl-4,4′-dihydroxydiphenylmethane (1.81 g, 5.74 mmol)in dry dimethylformamide (230 ml) was added dropwise via syringe pump toa stirred suspension of K₂CO₃ (7.92 g, 57.4 mmol) in drydimethylformamide (340 ml) at 80° C. over a period of 3 h. Afterstirring a further 4.5 h at 80° C. and 12 h at room temperature thereaction mixture was filtered and the dimethylformamide removed invacuo. The product was purified by chromatography on silica gel elutingwith 1% methanol/dichloromethane to give the title compound (0.47 g, 0.6mmol, 10.5%).

NMR (CDCl₃) δ 1.58-1.95 (m, 12H), 3.82 (s, 16H), 4.05 (t, J 8.0, 8H),6.82 (d, J 8.0, 4H), 7.16 (dd, J 8.0, 1.0, 4H), 7.58 (d, J 1.0, 4H).

1,7,21,27-tetraoxa-9,17,29,37-tetracarboxy[7.1.7.1]paracyclophane (17)

To a suspension of1,7,21,27-tetraoxa-9,17,29,37-tetra(methoxycarbonyl)[7.1.7.1]paracyclophane(0.47 g, 0.612 mmol) in methanol-water (3:1, 40 ml) was added solidsodium hydroxide (0.49 g (12.2 mmol). The resultant mixture was heatedto reflux for 1 h, then tetrehydrofuran (5 ml) was added and the mixtureagain heated to reflux for 2 h. The solvent volume was reduced by halfin vacuo and insoluble material removed by filtration. The filtrate wasacidified with conc hydrochloric acid and the resultant precipitatefiltered dried, and further washed with methanol-water before drying togive the title compound (160 mg, 0.22 mmol, 45%).

MS (EI) m/z 711 (M−H)⁻, ¹H NMR (DMSO) δ 1.51 (m, 4H), 1.68 (m, 8H), 3.81(s, 4H), 3.99 (t, J 5.6, 8H), 6.96 (m, 4H), 7.20 (m, 4H), 7.43 (m, 4H),12.25 (bs, 4H), ¹³C NMR (DMSO) δ 21.57, 27.81, 38.72, 68.47, 113.99,121.77, 130.28, 132.60, 133.21, 155.61, 167.27, I.R. (KBr) 1743 cm⁻¹.

In a Similar Manner were PreparedB2:1,10,24,33-tetraoxa-12,20,35,43-tetracarboxy-[2.2.1.2.2.1]paracyclophane,compound 18, starting from 3,3′-dicarboxymethyl-4,4′-dihydroxydiphenylmethane and α,α′-dibromo-p-xylene.

MS (EI) m/z 779 (M−H)⁻, ¹H NMR (DMSO) δ 3.75 (s, 4H), 5.15 (s, 8H), 6.91(d, J 8.58, 4H), 7.20 (m, 4H), 7.32 (m, 8H), 7.49 (m, 4H), 12.40 (bs,4H), ¹³C NMR (DMSO) δ 68.69, 84.57, 114.52, 126.54, 129.98, 132.32,133.81, 136.28, 154.60, 167.50, I.R. (KBr) 1733 cm⁻¹.

B3: Compound 25 starting from3,3′-dicarboxymethyl-4,4′-dihydroxydiphenyl methane and2,6-(dibromomethyl)naphthalene (Golden, J. H., J. Chem. Soc. 1961,3741).

MS (EI) m/z 879 (M−H)⁻, ¹H NMR (DMSO) ? 3.76 (s, 4H), 5.30 (s, 8H), 6.90(m, 4H), 7.19 (m, 4H), 7.49 (m, 8H), 7.74 (m, 4H), 7.83 (m, 4H), 12.60(bs, 4H), ¹³C NMR (DMSO) ??69.21, 83.84, 114.73, 121.88, 125.25, 125.37,127.96, 130.34, 132.04, 132.67, 133.97, 135.03, 154.91, 167.52, I.R.(KBr) 1731 cm⁻¹.

EXAMPLE 3

Compound 24:cyclo[(1-4)-α-L-rhamnopyranosyl-(1-4)-α-D-mannopyranosyl]-tetraoside.

The synthesis of this cyclic octasaccharide is described by Ashton etal. in Chem. Eur. J. 1996, 2, 580-591.

EXAMPLE 4 Complexation of Rocuronium Bromide by Chemical Chelators

All the ¹H spectra (303 K) were recorded at 400.13 MHz with 128 scans,sw=12 ppm, TD=32 k and zero filled to 64 k real points in processing.All experiments were measured at 303K.

Determination of Stoichiometry

Stock solutions of rocuronium bromide and γ-cyclodextrin (15) wereprepared, both with a concentration of 6.02 mM. From these, sixteensolutions were prepared (with the mole % of rocuronium ranging from0-100) by taking aliquots of 0-800 μl of the rocuronium bromidesolution. Aliquots of the γ-cyclodextrin solution, 800-0 μl were addedto make the solution a total volume of 800 μl and 100 mole % (i.e. 6.02mM of [rocuronium bromide+γ-cyclodextrin]). ¹H-NMR spectra were recordedas described above.

If the chemical shift change of H_(9α) in rocuronium bromide is definedas Δδ then a plot of [Δδ*(mole % rocuronium bromide)] vs. [mole %rocuronium bromide] gives a so called Job Plot by the method ofcontinuous variation (Connors K. A.: Binding constants, The measurementof Molecular Complex Stability; Wiley-Interscience; New York, 1987, pp24-28). The maximum in this plot is indicative for the stoichiometry ofthe complex. The Job Plot for the rocuronium bromide/γ-cyclodextrincomplex has a maximum at 50 mole % rocuronium, indicating thatrocuronium bromide and γ-cyclodextrin form a 1:1 complex.

Determination of the Association Constants

A stock solution of rocuronium bromide: 0.821 mM in D₂O was prepared.Stock solutions of β-cyclodextrin (9) and γ-cyclodextrin (15), both withconcentrations of 13.1, 6.57, 1.64, and 0.411 mM in D₂O, were prepared.Aliquots of 50-400 μl of these solutions were then removed and made upto 400 μl with D₂O (where required) and mixed with 400 μl of therocuronium bromide solution. To extend the data range for γ-cyclodextrin(15) experiments to higher cyclodextrin concentrations three additionalsolutions were prepared: 16.4, 24.6, 32.8 mM in 400 μl D₂O. As before,these solutions were mixed with 400 μl of the rocuronium bromidesolution.

¹H NMR spectra were recorded as described above.

The association constants of the complexes were derived from the plotsof proton chemical shift changes of the cyclodextrin and/or rocuroniumbromide signals (Δδ) versus the mole % cyclodextrin, using a curvefitting method (Loukas Y. L., J. Pharm. Pharmacol. 1997, 49, 941; BissonA. P., et al. Chem. Eur. J. 1998, 4, 845). The association constants arelisted in Table are listed in Table A.

TABLE A Association constants of 1:1 complex of rocuronium bromide andcyclic host compounds (K_(a), M⁻¹), determined by NMR spectroscopy at303 K. Chemical chelator Association constant (K_(a), M⁻¹)β-cyclodextrin (9) 3,100-3,900* γ-cyclodextrin (15) 10,000-20,400#*protons of CH₃-19 [rocuronium] and H₃ [β-cyclodextrin (9)] measured.#protons of H_(9α) [rocuronium] and H_(3,5) [γ-cyclodextrin (15)measured.

EXAMPLE 5 Reversal of Neuromuscular Blockade In Vivo AnaesthetizedGuinea Pig

Male Dunkin-Hartley guinea pigs (bodyweight: 600-900 g) wereanaesthetized by i.p. administration of 10 mg/kg pentobarbitone and 1000mg/kg urethane. After tracheotomy, the animals were artificiallyventilated using a Harvard small animal ventilator. A catheter wasplaced into the carotid artery for continuous monitoring of arterialblood pressure and the taking of blood samples for blood gas analysis.Heart rate was derived from the blood pressure signal. The sciatic nervewas stimulated (rectangular pulses of 0.5 ms duration at 10 s (0.1 Hz)intervals at a supramaximal voltage, using a Grass S88 Stimulator) andthe force of M. gastrocnemius contractions was measured using a GrassFT03 force-displacement transducer. Contractions, blood pressure andheart rate were recorded on a multichannel Grass 7D recorder. Catheterswere placed in both jugular veins. One catheter was used for thecontinuous infusion of a neuromuscular blocking agent. The infusion rateof the neuromuscular blocking agent was increased until a steady-stateblock of 85-90% was obtained. The other catheter was used foradministration of increasing doses of the reversal agent. Duringcontinuous infusion of the neuromuscular blocking agent, single doses ofincreasing concentration of reversal agent were given. At the end of theexperiment, the to measured force of muscle contractions was plottedagainst the concentration of reversal agent, and using regressionanalysis techniques, the 50% reversal concentration was calculated.

Results for the reversal of the neuromuscular block, induced by themuscle relaxants rocuronium bromide (Roc), vecuronium bromide (Vec),pancuronium bromide (Pan), mivacurium chloride (Miv), atracuriumbesilate (Atr), cis-atracurium (Cis-Atr), tubocurarine chloride (T-C),suxamethonium chloride (Sux; succinylcholine) and rapacuronium bromide(Rap; Org 9487), by means of a series of α-, β- and γ-cyclodextrins(compounds 1-16) and modified cyclodextrins are presented in Table I.The results demonstrate that the action of each of the neuromuscularblocking agents can be reversed by intravenous administration of acyclodextrin derivative.

TABLE I Dose (ED₅₀, μmol · kg⁻¹) producing 50% reversal of steady-stateneuromuscular block in anaesthetized guinea pigs. Compound* Roc Vec PanMiv Atr Cis-Atr T-C Sux Rap 1: α-cyclodextrin (α-CD) 1575 (2)  2610 (1) 2: carboxymethyl-β-CD (DS = 3.5) 134 (2)  800 (1)  sodium salt 3:2-hydroxy-3-trimethylammonio 518 (2)  1400 (1)  propyl--β-CD (DS = 3.5)4: per 2,6-dimethyl--β-CD  70 (2) 1313 (4)  (DS = 12.6) 5:β-cyclodextrin-phosphate 280 (3)  sodium salt (DS = 3) 6:β-cyclodextrin-phosphate 120 (2)  sodium salt (DS = 8) 7:carboxymethyl-β-CD (DS = 3-3.5) 139 (3)  8: carboxyethyl-β-CD (DS = 3)42 (3) 103 (2)  479 (2) 408 (2) 412 (2) 9: β-cyclodextrin (β-CD) 20 (3)636 (1)  1050 (2)  358 (3) 10: 2-hydroxypropyl-β-CD 33 (3) 1598 (4)  11:γ-cyclodextrin-phospate 64 (3) 67 (3) 197 (2) 292 (2) 113 (2) 204 (2)160 (2) 160 (2)  80 (3) sodium salt (DS = 3) 12: γ-cyclodextrin-phospate52 (2) sodium salt (DS = 7) 13: carboxymethyl-γ-CD (DS = 3.2) 25 (4) 30(3) 641 (4) 964 (3) 990 (2) 893 (2) 431 (2) 999 (2) 227 (2) 14:carboxyethyl-γ-CD (DS = 3.8)  7 (3) 25 (3) 141 (3) 399 (2) 421 (2) 1558(3)  294 (2) 1294 (2)   43 (3) 15: γ-cyclodextrin (γ-CD)  4 (3) 75 (4)186 (4) 1690 (2)  2793 (4)  1710 (2)  630 (2) 1189 (2)   57 (3) 16:2-hydroxypropyl-γ-CD (DS = 4) 12 (3) 123 (3)  440 (3) 1674 (2)  828 (2)878 (2) 909 (2) 1340 (2)  134 (3) ED₅₀ values are the mean of a numberof experiments; the number is shown in parenthesis.

EXAMPLE 6 Reversal of Neuromuscular Block In Vitro Isolated MouseHemidiaphragm Preparation

Hemidiaphragms, with phrenic nerves attached, were removed fromeuthanized male mice (Institute of Cancer Research; bodyweight: 20-60g). The preparations were mounted on a tissue holder and placed in atissue bath filled with a modified Krebs-Henseleit solution(composition: 118 mM NaCl, 30 mM NaHCO₃, 5 mM KCl, 1 mM KH₂PO₄, 1 mMMgSO₄, 30 mM glucose and 2.5 mM CaCl₂) at 37° C. and bubbled with 95%oxygen and 5% carbondioxide. One end of the preparation was connectedwith a siliconized silk suture to a Grass FT03 force-displacementtransducer. An initial force of 10 mN was applied. The phrenic nerveswas placed on a bipolar platinum electrode and was stimulated withrectangular pulses of 0.2 ms duration at 20 s (0.05 Hz) intervals at asupramaximal voltage, using a Grass S88 Stimulator. Contractions wererecorded on a four channel Grass 79D recorder.

After the development of stable contractions, an appropriate single doseof a neuromuscular blocking agent was added to each bath to produceinhibition of contractions to approximately 5-10% of baseline valuesafter 20 min contact time (this concentration was found to be 3.1 μM forrocuronium bromide). Increasing amounts of reversal agent were thenadministered into the bath at intervals of 10 min. The % maximumreversal was established. At the end of the experiment, the measuredmuscle contractions force was plotted against the concentration ofreversal agent, and using regression analysis techniques, the 50%reversal concentration was calculated.

After induction of neuromuscular block by rocuronium, the % maximumreversal produced by the addition of a number of γ-cyclodextrinderivatives (compounds 11-16), a cyclic octasaccharide comprising 4rhamnosyl-manno-pyranosyl units (compound 24), or a number ofparacyclophane derivatives (compounds 17-21 and 23) are presented inTable II. The results demonstrate that the neuromuscular blocking actionof rocuronium can be efficiently blocked by chemical chelators ofvariable structure, i.e. by the γ-cyclodextrins 13, 14 and 15, by acyclic oligosaccharide composed of rhamnose and mannose (24), and byaction of the cyclophanes 18 and 23.

TABLE II Mouse hemidiaphragm: maximum reversal (%) 3.1 μM Compoundrocuronium¹ CYCLODEXTRINS 11: γ-cyclodextrin-phospate sodium salt (DS =3) 34 (360) 12: γ-cyclodextrin-phospate sodium salt (DS = 7) 17 (360)13: carboxymethyl-γ-CD (DS = 3.2) 88 (180) 14: carboxyethyl-γ-CD (DS =3.8) 87 (144) 15: γ-cyclodextrin (γ-CD) 94 (144) 16:2-hydroxypropyl-γ-CD (DS = 4) 71 (360) CYCLIC OLIGOSACCHARIDE 24:cyclo[(1-4)-α-L-rhamnopyranosyl-(1-4)-α-D- 86 (288)mannopyranosyl]tetraoside CYCLOPHANES 17:1,7,21,27-tetraoxa-9,17,29,37-tetracarboxy- 11 (360)[7.1.7.1]paracyclophane 18:1,10,24,33-tetraoxa-12,20,35,43-tetracarboxy-- 61 (288)[2.2.1.2.2.1]paracyclophane 19: N,N′,N″,N′″-tetrakis(carboxyacetyl)- 2.4(108)  1,7,21,27-tetraaza[7.1.7.1] paracyclophane 20:N,N′,N″,N′″-tetrakis(4-carboxybutyryl)-1,7,21, 8.7 (72)  27-tetraaza[7.1.7.1] paracyclophane 21:N,N′,N″,N′″-tetrakis(3-carboxypropionyl)-  0 (360)1,7,21,27-tetraaza[7.1.7.1] para cyclophane 23:N,N′,N″,N′″-tetrakis(3-carboxypropionyl)- 90 (58) 3,4,5,6,7,8,26,27,28,29,30,31-dodecahydro-1,10,24,33-tetraaza[2.2.1.2.2.1]paracyclophane ¹concentration ofchemical chelator at maximum reversal in μM in parenthesis;

EXAMPLE 7 Calixarene Derivatives

4-Sulfonic calix[6]arene and 4-sulfonic calix[8]arene were obtained fromAldrich.

Reversal of neuromuscular block in vivo, induced by rocuronium bromide,was carried out as described in Example 5. The dose (ED₅₀) of thecalixarene derivative producing 50% reversal of steady-stateneuromuscular block in the anaesthetized guinea pig was found to be 5.1μmol/kg for the 4-sulfonic calix[6]arene and 34 μmol/kg for the4-sulfonic calix[8]arene.

Reversal of neuromuscular block in vitro was carried out using the mousehemidiaphragm preparation as described in Example 6. After induction ofneuromuscular block (95% block) with rocuronium bromide (3.6 μM in thebath) maximum reversal of 124% and 120% were recorded for 4-sulfoniccalix[8]arene and the 4-sulfonic calix[6]arene, respectively, while the50% reversal concentrations were found to be 36 μM and 34 μM.

1-11. (canceled)
 12. A method for reversal of drug-induced neuromuscularblock in a patient caused by clinically-used neuromuscular blockingagent which acts by reversible binding to the acetylcholine receptor,comprising parenterally administering to said patient an effectiveamount of a chemical chelator capable of forming a guest-host complexwith the neuromuscular blocking agent, wherein the association constantof said guest-host in an aqueous medium at ambient temperature is higherthan 10.000 M-1.
 13. The method according to claim 12, wherein theneuromuscular blocking agent is selected from the group consisting ofrocuronium, vecuronium, pancuronium, rapacuronium, mivacurium,(cis)atracurium and tubocurarine.
 14. The method according to claim 12,wherein the chemical chelator is selected from the group consisting ofcyclic oligosaccharides and cyclophanes.
 15. The method according toclaim 12, wherein the chemical chelator is gamma-cyclodextrin or aderivative thereof.
 16. The method according to claim 12, wherein theneuromuscular blocking agent is selected from the group consisting ofrocuronium and vecuronium and the chemical chelator isgamma-cyclodextrin or a derivative thereof.